Subzero hydrogen-deuterium exchange CE-MS

We have performed in solution hydrogen exchange (HDX) on peptide standards and bovine hemoglobin with subsequent quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for the benchmarking of a lab built HDX-MS platform. Though neutral coated capillaries that eliminate electroosmotic flow provide fast separations with peak capaci-ties surpassing 200, uncoated capillaries with high electroosmotic flow achieve half as much peak ca-pacity with even faster separations but 30% higher deuterium retention for angiotensin II peptide standard. The data obtained from two different separation conditions on peptic digests of bovine he-moglobin shows strong agreement for relative deuterium uptake between methods and provides data amenable to software such as MS-Studio. Processed data from denatured hemoglobin vs the longest time point in this study (50,000s) also shows agreement with subunit interaction sites determined by crystallographic methods.